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United States Patent | 5,316,917 |
Roe | May 31, 1994 |
Disclosed is a method of assaying free L-carnitine and total short-chain acylcarnitine in blood. The method uses essentially protein-free samples of plasma or serum and comprises (a) obtaining a sample of ultrafiltrated plasma or serum, (b) converting essentially all carnitine to acetylcarnitine using an enzymatic reaction, (c) converting essentially all free coenzyme A liberated in the enzymatic reaction of (b) to thiophenylate, (d) evaluating the sample spectrophotometrically to determine the amount of thiophenylate present, (e) obtaining a fresh sample of ultrafiltrated plasma or serum and hydrolyzing the sample to convert essentially all short-chain acylcarnitine to carnitine, and (f) repeating steps (b) through (d). All steps following the ultrafiltration of the sample are carried out within a pre-programmed, automated centrifugal analyzer. The use of this method to measure only free L-carnitine or only total short-chain acylcarnitine in plasma or serum samples is also disclosed, as is a kit for carrying out the automated analysis of free L-carnitine and total short-chain acylcarnitine.
Inventors: | Roe; Diane S. (Chapel Hill, NC) |
Assignee: | Duke University (Durham, NC) |
Appl. No.: | 971888 |
Filed: | November 4, 1992 |
Current U.S. Class: | 435/15; 435/4; 435/7.91; 435/14; 435/117; 435/128; 435/188; 435/190; 435/252.8; 560/145 |
Intern'l Class: | C12Q 001/48; C12Q 001/54; C12N 009/02; A61K 043/00 |
Field of Search: | 435/15,188,190,117,128,4,7.91,252.8 424/1.1 560/145 |
4221869 | Sep., 1980 | Vandecasteele et al. | 435/875. |
5028538 | Jul., 1991 | Seim et al. | 435/128. |
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TABLE 1 __________________________________________________________________________ TOTAL SHORT-CHAIN ACYL FREE L-CARNITINE CARNITINE __________________________________________________________________________ GENERAL GENERAL Measurement Mode: ABS Measurement Mode: ABS Rxn Mode: P-I-SR1-A Rxn Mode: S-I-W-SR1-I-R1-I-SR2-A Calibration Mode: Lin Reg Calibration Mode: Lin Reg Reagent Blank: Reag/Dil Reagent Blank: Reag/Dil Wavelength: 412 Wavelength: 412 Temperature: 37.degree. C. Temperature: 37.degree. C. Unit: .mu.mol/L Unit: .mu.mol/L ANALYSIS ANALYSIS P Sample Vol: 25 .mu.L S Sample Volume: 40 .mu.L Diluent Name: H.sub.2 O Diluent Name: KOH Vol: 5 .mu.L Vol: 5 .mu.L Cleaner Cycle: off Cleaner Cycle: off Reagent Volume 30 .mu.L I Incubation: 150 s I Incubation: 480 s M1: 20 s M: No M2: 140 s M: No SR1 Start Reagl: 10 .mu.L W Wait Time: 480 s Diluent Name: H.sub.2 O SR1 Start Reagl: 5 .mu.L Vol: 5 .mu.L Diluent Name: H.sub.2 O Wet Cycle: on Vol: 0 .mu.L A Readings: Wet Cycle: off First: 1.0 s I Incubation: 240 s Number: 27 M: No Interval: 20 s M: No M: No R1 Reag 1: 30 .mu.L M: No I Incubation: 150 s M1: 20 CALCULATION M2: 140 s Reaction Direction Increase SR2 Start Reag 2: 10 .mu.L Check: On Diluent Name: H.sub.2 O Calculation Step A: Kinetic Vol: 5 .mu.L First: 1 Wet Cycle: On Last: 27 A Readings: First: 1.0 s CALIBRATION Number: 27 Cal. Interval: Each Run Interval: 20 s Number of Stds: 5 M: No Position of Stdl: 1 M: No STD1: 10.0 STD2: 20.0 STD3: 30.0 STD4: 40.0 CALCULATION STD5: 100.0 Reaction Direction: Increase Check: On Calculation Step A: Kinetic First: 1 Last: 27 CALIBRATION Cal. Interval: Each Run Number of Stds: 5 Position of Std1: 1 STD1: 20.0 STD2: 40.0 STD3: 60.0 STD4: 80.0 STD5: 200.0 __________________________________________________________________________